The stability of proteins is generally a problem in pharmaceutical industry. It has often been solved by drying the protein in various drying processes, such as freeze-drying. The protein has thereafter been distributed and stored in dried form. The solution before drying or freeze-drying, the dried material and the reconstituted product should all be stable, to avoid a substantial loss of activity in the drying process, as well as during storage or handling. The freeze-drying process is a costly and time consuming process step, and it would be of great advantage if this step could be avoided, when preparing a commercial product. Furthermore, the patient necessarily has to reconstitute the dried protein in a solvent before use, which could be inconvenient for the patient.
Haemophilia is an inherited disease which has been known for centuries but it is only within the last three decades that it has been possible to differentiate between the various forms; haemophilia A, haemophilia B and haemophilia C. Haemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10 000 live-born males. The disease is caused by strongly decreased level or absence of biologically active coagulation factor VIII (antihaemophilic factor) which is a protein normally present in plasma. The clinical manifestation of haemophilia A is a strong bleeding tendency and before treatment with factor VIII concentrates was introduced, the mean age of those patients was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has improved the situation for treatment of haemophilia patients considerably and given them possibility to live a normal life.
Therapeutic factor VIII concentrates have until now been prepared by fractionation of plasma. However, there are now methods available for production of factor VIII in cell culture using recombinant DNA techniques as reported in e.g. J Gitschier et al. Nature 312, p. 330-37, 1984 and EP-A-160 457.
Factor VIII concentrates derived from human plasma contain several fragmented fully active factor VIII forms (Andersson et al, Proc. NatI. Acad. Sci. USA, Vol 83, p. 2979-83, May 1986). The smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 kDa and 80 kDa held together by a metal ion bridge. Reference is here made to EP-A-1 97 901.
Kabi Pharmacia has developed a recombinant factor VIII product which corresponds to the 170 kDa plasma factor VIII form in therapeutic factor VIII concentrates. The truncated recombinant factor VIII molecule is termed r-VIII SQ and is produced by Chinese Hamster Ovary (CHO) cells in a cell culture process in serum free medium at finite passage.
The specific activity of r-VIII SQ is about 15 000 IU VIII:C per mg protein.
Recombinant factor VIII SQ is indicated for treatment of classical haemophilia. The dosage is similar to the dosage of the plasma factor VIII concentrates.
The structure and biochemistry of recombinant factor VIII-products in general have been described by Kaufman in Tibtech, Vol 9,1991 and Hematology, 63, p. 155-65, 1991. The structure and biochemistry of r-VIII SQ have been descrbed in WO-A-91/09122.
Factor VIII which has been fractionated from plasma is normally sold as freeze-dried powder which should be reconstituted with water.
A formulation with a low amount of protein will generally loose activity during purification, sterile manufacturing, in the package and during administration. This problem is usually solved by the addition of human albumin which reduces the loss of the active protein considerably. Human albumin functions as a general stabiliser during purificationa, sterile manufacturing and freeze-drying (see review by Wang et al., J. of Parenteral Sci. and Tech. Vol 42, Number 2S, supplement. 1988). The use of albumin for stabilisation of factor VIII is known and is currently used in all highly purified factor VIII products on the market.
However, it is desirable to avoid addition of human albumin to a therapeutic protein manufactured by recombinant DNA technology. In addition, the use of human albumin as a formulation excipient often limits the use of many of the most powerful and sensitive analytical methods for protein characterisation.
Several solutions have been proposed for stabilisation of different proteins. Thus, EP 35 204 (Cutter) discloses a method for imparting thermal stability to a protein composition in the presence of a polyol. Furthermore, WO-A-89/09614 (Genentech) discloses a stabilised formulation of human growth hormone comprising glycine, mannitol and a buffer is disclosed and in a preferred embodiment a non-ionic surfactant such as polysorbate 80 is added. The non-ionic surfactant is added for reduced aggregation and denaturation. The formulation has an increased stability in a freeze-dried formulation and upon reconstitution. Also, U.S. Pat. No. 4,783,441 (Hoechst) discloses an aqueous solution comprising a protein, such as insulin and a surface active substance.
EP 77 870 (Green Cross) discloses the addition of amino acids, monosaccharides, oligo-saceharides or sugar alcohols or hydrocarbon carboxylic acid to improve stability of a solution containing factor VIII. EP 117 064 (Green Cross) discloses the addition of sugar alcohol or disaccharides to an aqueous solution of factor VIII for increasing the stability during heat treatment.
WO-A-91/10439 (Octapharma) claims stable injectable solution of factor VIII or factor IX which comprises a disaccharide, preferably saccarose and one or more amino acids and EP 315 968 and EP 314 095 (Rorer) claim stable formulations of factor VIII with different ionic strength.
U.S. Pat. No. 4,727,027 (Diamond Scientific) is directed to a method for photochemical decontamination of aqueous compositions containing biologically active proteins derinved from blood or blood components, for minimizing loss in activity. The method comprises adding at least one furocoumarin to the composition and irradiating the obtained composition with ultraviolet (UV) light. Prior to the irradiation, the oxygen concentration of the aqueous composition can be reduced to inhibit denaturation. This can be achieved, e.g. by addition of oxygen scavengers, albumins and/or enzyme systems and/or flushing with an inert gas. Solutions containing factor VIII were flushed with argon with or without ascorbate for up to 6 hours. U.S. Pat. No. 4,727,027 is silent about storage of solutions for a prolonged time, as well as the possible effect of reduced oxygen concentration on factor VIII activity in such storage.
EP-A-0 212 040 (Immuno) relates to virus inactivation of factor VIII by heating of dry substance in an oxygen reduced environment. The heat treatment is carried out in the absence of stabilisers, since the latter protects also the viruses thereby reducing the efficiency of the treatment. Tests are carried out at 90.degree. C. for 30 hours. EP-A-0 212 040 is silent about the problem of poor stability of aqueous solutions containing factor VIII, which generally is a much more difficult problem to overcome than poor stability of dry products. This is because chemical changes, e.g. hydrolysis and deamidation, are much more pronounced in a solution than in the dry state.
Proteins are different with regard to physio-chemical properties. When preparing a pharmaceutical preparation which should be physiologically acceptable, and stable for a long time, consideration can not only be taken to the properties of the protein but also other aspects must be considered. Examples of the latter, are the industrial manufacture, as well as ease of handling and safety for the patient. The consequences of these aspects are not predictable when testing different formulations and there is often a unique solution for each protein.
It would facilitate the use and manufacture of factor VIII if the protein could be formulated and distributed to the patient as a stable solution without the addition of albumin and with a prolonged storage life. Also for the patient such a solution would facilitate the handling of the final drug product. The patient could thus inject the content of the final drug product directly without reconstitution.
Aqueous solutions containing oxygen-sensitive chemical compounds including drugs other than proteins, could be deoxygenated as follows:
Water for injection is bubbled with nitrogen to reduce the concentration of oxygen. PA1 The components are dissolved and the solution is bubbled with nitrogen and thereafter kept under a nitrogen blanket. During filling, the bottles are flushed with nitrogen gas and the bottles are closed under a stream of nitrogen. PA1 i) 10-100000 IU/ml of recombinant coagulation factor VIII PA1 ii) at least 0.01 mg/ml of a polyoxyethylene sorbitan fatty acid ester PA1 iii) sodium chloride, preferably in an amount of more than 0.1M. PA1 iv) calcium salt, such as calcium chloride or calcium gluconate, preferably in an amount of more than 0.5 mM. PA1 v) an amino acid, such as L-histidine, in an amount of more than 1 mM. PA1 vi) a mono- or disaccharide or a sugar alcohol, preferably sucrose or mannitol
It is, however, not possible to deoxygenate a protein solution by bubbling the solution with a gas. Protein solutions will foam heavily and many protein drugs, such as coagulation factor VIII, will denature if exposed to such a treatment. Therefore, it has never been suggested earlier that an aqueous solution containing coagulation factor VIII should be stored under an inert gas such as nitrogen.